Oocytes Derived from Ovarian Culture Initially Containing No Oocytes

ABSTRACT

Ovarian germ-line-competent embryonic stem cells (GLC-ESC) are cultured, either in the presence or absence of a compound having estrogenic activity. The GLC-ESC are either collected prior to specific commitment or are permitted to remain in the culture medium for a time sufficient to develop into oocytes, and the oocytes may be fertilized by adding sperm to the culture medium. The fertilized oocytes may be permitted to develop into embryos, which may be transferred into the uterus of an adult human female or frozen for later use. The invention provides a method for obtaining by in vitro fertilization an embryo that is genetically related to a human female who is not producing oocytes.

This application is a continuation of pending U.S. patent applicationSer. No. 11/473,910, filed Jun. 23, 2006, which application has beenallowed and is to issue as U.S. Pat. No. 8,232,077 on Jul. 31, 2012,which application claims priority from U.S. Provisional PatentApplication Ser. No. 60/696,952, filed on Jul. 6, 2005.

FIELD OF THE INVENTION

This invention pertains to the field of culturing cells and inparticular to the field of culturing human ovarian cells and to thefield of utilizing oocytes cultured from these cells for fertilizationand implantation into a human uterus.

BACKGROUND OF THE INVENTION

The belief that all primary follicles in adult mammalian females wereformed during the fetal period has persisted for over fifty years,primarily due to the diminution in the number of primary follicles thatoccurs with age. Recent studies, however, have brought this belief intoquestion.

Bukovsky, et al, Am. J. Reprod. Immunol., 33:323-340 (1995), reportedthat the ovarian surface epithelium (OSE) is a source of germ cells inadult human females.

Bukovsky, et al, Reprod. Biol. Endocrinol., 2:20 (2004) reported thatnew primary follicles are formed by assembly of oocytes with nests ofprimitive granulosa cells in the human ovarian cortex during adulthood.

These studies have suggested that the hypothesis that all primaryfollicles in adult human females were formed during the fetal period isincorrect. Rather, these studies indicate that primary follicles thatwere formed during the fetal period may persist for some time, perhapsthrough childhood, and then starting with menarche, new cohorts ofprimary follicles replace the fetal follicles that undergo atresia.Moreover, it is suggested that during each successive menstrual cycleuntil about the age of 38, fresh follicles are produced that replaceolder follicles. At about the age of 38±2 years, formation of newprimary follicles ceases, possibly due to the onset of immune senescence(certain immune system-related cells appear to be required for theformation of new germ cells from the OSE), and the aging folliclesaccumulate genetic alterations until exhausted at the onset of naturalmenopause.

In recent years, women in developed countries have an increased tendencyto delay having a first child. Reasons for waiting to start a familyinclude the desire to wait until the family has financial security andthe desire of women to commit to their marriages and to their careersbefore focusing their energy on children. According to the NationalCenter for Health Statistics, the birth rate for women aged 40-45 yearsrose 20% between 1990 and 1995, and increased 74% during 1981-95. Therising birth rate along with the increasing number of women in this agegroup means that there were more babies born in 1995 to mothers in their40s than in any year since 1966.

A significant problem that occurs due to delayed parenting is that oftenwomen over the age of 40 have a greatly decreased chance of becomingpregnant. Much of this difficulty stems from a decrease in theavailability of oocytes that are available for fertilization.Additionally, women below the age of 40 that have premature ovarianfailure are unable to become pregnant due to the lack of production ofoocytes.

A woman who desires to become pregnant but who does not produce her ownoocytes may elect to undergo a procedure referred to as in-vitrofertilization (IVF) in which eggs donated from another woman arefertilized in vitro and then one or two of the resultant developingembryos are implanted into her uterus. Of course, the resultant childfrom this procedure will be genetically unrelated to the womanundergoing this procedure. At the present time, there are no proceduresavailable by which a woman who does not produce oocytes is able toproduce a child who is genetically related to her.

An important need exists for the development of methodologies that willpermit a woman who does not produce oocytes that have the potential todevelop into an offspring who is genetically related to her.

DESCRIPTION OF THE INVENTION

It has been unexpectedly discovered that that cultures of adult ovarianstem cells are capable of differentiating into oocytes and mature eggs.These oocytes may be fertilized in vitro and then implanted into theuterus of an adult human female. The fertilized oocytes are capable offurther developing into human embryos.

In one embodiment, the invention is a method for obtaining human oocytesfrom culture. According to this embodiment of the inventiongerm-line-competent embryonic stem cells (GLC-ESC cells), which areovarian surface epithelial (OSE) cells or activated mesenchymal tunicaalbuginea (TA) cells, are cultured for a time sufficient to produceoocytes in the culture medium. Activated mesenchymal TA cells are thosethat express cytokeratin. The oocytes thus produced may be used for theautologous or allogeneic treatment of ovarian infertility. For thatpurpose, the oocytes may be fertilized in vitro and then permitted todevelop into embryos, which may be collected and utilized, such as forintrauterine implantation, for the treatment of ovarian sterility. ifdesired,, the oocytes may be frozen, before or after fertilization, andutilized at some time in the future.

In another embodiment, the invention is a method for obtainingfertilized oocytes. According to this embodiment of the inventiongerm-line-competent embryonic stem cells (GLC-ESC cells), which areovarian surface epithelial (OSE) cells or activated mesenchymal tunicaalbuginca (TA) cells, arc cultured for a time sufficient to produceoocytes in the culture medium. The oocytes are fertilized in vitro andthen permitted to develop into embryos, which may be collected andutilized, either fresh or frozen and thawed, for intrauterineimplantation for the treatment of ovarian sterility.

In another embodiment, the invention is a method for in-vitrofertilization. According to this embodiment of the invention,germ-line-competent embryonic stem cells (GLC-ESC cells), which areovarian surface epithelial (OSE) cells or activated mesenchymal tunicaalbuginea (TA) cells, are cultured for a time sufficient to produceoocytes in the culture medium. The oocytes are fertilized in vitro andthen permitted to develop into embryos, which may be collected andtransferred, either fresh or following freezing and thawing, into theuterus of a human adult female.

In another embodiment, the invention is a method for producing a humanembryo that is genetically related to a donor female human. According tothis embodiment of the invention, germ-line-competent embryonic stemcells (GLC-ESC cells), which are ovarian surface epithelial (OSE) cellsor activated mesenchymal tunica albuginea (TA) cells, are obtained fromthe ovary of an adult female human. The cells are cultured for a timesufficient to produce oocytes in the culture medium. The oocytes arefertilized in vitro and then permitted to develop into embryos, whichembryo is genetically related to the female human. The embryo thusobtained may be collected and transferred, either fresh or followingfreezing and thawing, into the uterus of the female human.

The culture medium for this embodiment of the invention may or may notcontain a chemical compound that has estrogenic activity. One example ofsuch a chemical compound is phenol red. GLC-ESC cells that are culturedin accordance with the method of the invention in the presence of achemical compound, such as phenol red, that has estrogenic activity,typically produce oocytes starting at about 4 to 6 days after initiationof the culture. It is conceived that an effect of estrogen is to drivethe GLC-ESC cells to develop into oocytes.

Alternatively, the culture medium may be devoid of a chemical compoundthat has estrogenic activity prior to placing cells in the medium. Inthis case, it has been surprisingly discovered that oocytes will alsodevelop. Such development of oocytes, however, occurs later in time thanthat which occurs in culture medium containing a source of estrogenicactivity. Typically, oocytes are detected in culture medium lacking achemical compound after 10 to 12 days in culture. Although the inventorsdo not intend to be bound by theory, it is conceived that estrogen maybe required for the development of oocytes from GLC-ESC cells and that,in the case of a culture medium free of estrogenic activity, suchestrogenic activity may come from cells, such as fibroblasts, that oftenaccompany the GLC-ESC cells in culture.

The GLC-ESC cells may be obtained by any means by which such cells arecapable of being obtained. The cells preferably are removed, such as byscraping, from the surface of an ovary within a subject, such as duringa surgical or laparoscopic procedure or from a biopsy sample of anovary. The cells may also be removed, such as by scraping, from thesurface of an ovary that has been surgically removed from a patient.Suitable OSE cells may also be obtained, not only from the ovariansurface, but also from OSE crypts within the ovary, for example byscraping the stroma of dissected ovaries or by trypsinization of a smallovarian biopsy.

The GLC-ESC culture may be a primary or secondary culture. Primarycultures of ovarian tissue often include various types of cells,including granulosa cells, epithelial cells, and fibroblasts. Secondarycultures lack granulosa cells.

The ovaries from which the GLC-ESC cells are obtained may be from anadult woman of any age. The woman may be premenopausal, postmenopausal,or anovulatory. It has been discovered that OSE cells cover the ovariansurface more thoroughly in ovaries that are postmenopausal oranovulatory. Therefore, because of the relative ease of procuringsuitable GLC-ESC cells, such ovaries are preferred. However, if present,GLC-ESC cells from any ovary may be utilized.

In culture, in addition to their characteristic morphology, the oocytesmay also be identified by alkaline phosphatase activity. Zona pellucida(ZP) proteins and some ZP antigens, such as PSI meiotically expressedcarbohydrate antigen and heat-solubilized porcine zona (HSPZ) proteins,are more specific markers. The oocytes may also express CK18, a markerof the Balbiani body. Furthermore, oocytes also express the intermediatefilament vimentin, a protein which plays an important role in thematuration and fertilization of eggs.

The invention is further illustrated in the following non-limitingexamples.

Example 1 Production of Oocytes from Ovarian Culture Example 1aCollection of GLC-ESC Cells

All chemicals and consumables, except where specified otherwise, werepurchased from Sigma Chemical Co., St. Louis, Mo.. Cultured cells werecollected from ovaries associated with fresh hysterectomy/bilateralsalpingooophorectomy specimens. The surgery was performed for medicalindications, including chronic pelvic pain, uterine fibroids, and/oruterine bleeding (severe dysmenorrhea) not responding to conservativetreatment. The surface of intact ovaries was gently scraped in anaseptic laminar flow hood with a sterile stainless steel surgery knifeblade No. 21 (Becton Dickinson, AcuteCare, Franklin Lakes, N.J.). Thisprocedure was selected with an intention include OSE and some adjacentTA mesenchymal cells.

Example 1b Culture of GLC-ESC Cells

The cells of Example la were collected into sterile petri dishescontaining tissue culture medium supplemented with heat inactivated 20%fetal bovine serum (FBS; Gibco/BRL, Grand Island, N.Y.) and antibiotics(50 μg/ml gentamycin, 100 U/ml penicillin, and 100 μg/ml streptomycin).The tissue culture media utilized was either Dulbecco's Modified EagleMedium/Ham's F12, phenol red free (DMEM/F12; without estrogenic stimuli)or Dulbecco's Modified Eagle's Medium containing 25 mM HEPES, 4500 mg/Lglucose, and phenol red (DMEM-HG; with estrogenic stimuli). There was noother treatment imposed during the culture.

The cells were spun down (1000×g, 5 min, 24° C.), diluted in 0.75-1.5 mlof supplemented media, seeded in either 3 or 6 wells of a 24-well plate(250-350 μl per well) (Fisher Scientific, Pittsburgh, Pa.), and culturedin an humidified atmosphere with 5% CO2 at 37° C. The number of wellswas chosen by the size of ovaries. Cells collected from small ovarieswere seeded into 3 wells, and from larger ovaries into six wells. Allovaries involved in the experiment were anovulatory, as no corpora luteawere detected. The culture medium was changed once after 24 hours. Thisleft only adherent (viable) cells in culture, and eliminatednon-adherent cells and the majority of contaminating erythrocytes. Thecell cultures were monitored daily by phase contrast microscopy and livecells evaluated by immunohistochemistry after 5-6 days from the initialseeding. Viability of cells was apparent from their active movement,changes in shape, and movement of their nuclei. The number of adherentcells in a single well of 24-well plate ranged between ˜100 to 1000during the late culture period (day 5 or 6).

Example 1c Immunohistochemistry

The medium was aspirated from wells of the 24-well plate, and cells wereallowed to dry under a fan before placing them in a ventilated hoodovernight. The bottoms of the wells were dried in the upright positionand found macroscopically dry within a few seconds. The cells were fixedwith 96% ethanol for 5 minutes, allowed to dry again, and incubatedovernight (4° C.) with primary antibodies against zona pellucida (ZP)proteins: rabbit-anti heat solubilized porcine zona (HSPZ, 1:20) ormouse monoclonal PS1 antibody recognizing meiotically expressed ZPcarbohydrate antigen diluted 1:10 in phosphate buffered solution (PBS),pH 7.22. We also used mouse-anti human CK18, clone CY90, Sigma (diluted1:50), mouse antihuman CK5,6,8,17, clone MNF116 (DAKO Corporation,Carpinteria, Calif.) (diluted 1:50), and mouse antihuman vimentin, cloneV9 (DAKO Corporation) (diluted 1:50). As a control, an HLA-DR antibodywhich does not react with OSE cells, was used. We used an HLA-DRantibody not reacting with fibroblast, granulosa, epithelial, germ oroocyte cell types, in order to identify activated tissue macrophages,but did not find any such cells in day 5 or 6 cultures. After severalwashes in PBS (room temperature), the cells were incubated withperoxidase labeled corresponding secondary antibodies—goat anti-rabbitIgG, pre-absorbed with human serum (Jackson Immunoresearch, West Grove,Pa.), diluted 1:50; or swine anti-mouse IgG, diluted 1:50 and absorbedwith rat kidney homogenate to remove background. After additional washesin PBS (room temperature), the bound antibodies were visualized bydiaminobenzidine substrate, but without hematoxylin counterstain,covered with PBS, and images captured as described below. The cellsstained for CK18 or ZP were processed further for dual colorimmunohistochemistry to identify co-expression of other proteins, andvisualized with blue chromogen substrate. Finally, the washed cells werecovered with PBS containing 0.01% azide as a preservative.

Example 1d Image Processing

Two independent observers evaluated the live cells by phase contrast andsubsequent immunohistochemistry using an inverted microscope (NIKON,Nikon Inc., Instrument Division, Garden City, N.Y.) equipped with aDEI-470 CCD Video Camera System (Optronics Engineering, Goleta, Calif.)with detail enhancement. The video images were captured by CG-7 colorframe grabber (Scion Corporation, Frederick, Md.) supported by ScionImage public software developed at the National Institutes of Health(Wayne Rasband, NIH, Bethesda, Md.), and ported to Windows XP, 2002release (Microsoft Corporation, Redmond, Wash.). To obtain figurepanels, the captured video images were copied into Microsoft®Power-Point® 97 SR-2 (Microsoft Corporation). Each image (includingcontrols) was further copied into Microsoft Photo Editor 3.0 (MicrosoftCorporation), and blue saturation adjusted (brightness 70, contrast 70,gamma 0.30). In total, more than 100 images were captured and stored.

Example 1e 40 Year Old Female

Ovarian cells were collected from the ovary of a 40-year old woman asdescribed in Example 1a and cultured in DMEM-HG supplemented with 20%FBS and antibiotics as described in Example 1b. Initial examination ofthe cells at time of culture showed a uniform pattern of epithelial typecells, while no other cell types, including mesenchymal and granulosacells, were present.

Phase contrast microscopy, as described in Example 1d, revealed largecells exhibiting an oocyte phenotype in the OSE culture after 5 days.These cells reached 180 μm in diameter and showed a centrally located 40μm nucleus with nucleolus, and perinuclear accumulation of organelles.

Immunohistochemical staining of the cells, as described in Example 1c,showed diffuse CK immunoexpression with a preserved accumulation ofstaining around the cell nucleus. The perinuclear space also exhibitedenhanced staining for ZP proteins in the same cell. Controlimmunohistochemical staining using the anti-HLA antibody showed noreaction. Clusters of OSE cells with perinuclear ZP expression anddifferentiation into large cells resembling oocytes was also observed.

Example 1f 39 Year Old Woman

Ovarian tissue was collected from a 39 year-old woman as described inExample 1a. Prior to culture, a portion of the collected ovarian tissuewas passed through a cell strainer in order to ensure that mostly singlecells, and not cell sheets, were seeded in the culture medium.Unstrained and strained cells were cultured, as described in Example 1b,in medium containing phenol red (DMEM-HG; with estrogenic stimuli) or inmedium lacking phenol red (DMEM/F12; without estrogenic stimuli).

Except for the occasional mesenchymal cells of the fibroblast phenotype,there was no evidence of cell commitment until day 4 of culture. On day5 the cells collected from the ovarian surface and strained did notprogress beyond the state found on day 3, and no oocytes were detectedregardless of the presence or absence of phenol red. However, many cellswith an oocyte phenotype were found in mixed cultures, unstrained cells,in cultures containing phenol red. They showed moderate (100 μm) sizewithout zona pellucida and were accompanied by fibroblasts. Largeroocytes (120 μm) accompanied by fibroblasts showed a developing zonalayer.

The reason that oocytes were not found on day 5 culture (last day ofculture) of cells that had been passed through a cell strainer was notdetermined. It is conceived that the passage of the cells through thecell strainer prevented the passage of OSE cell sheets.

Because the culture was not continued beyond day 5, it is also conceivedthat it may require additional time for the development of oocytes fromcultures lacking sheets of OSE cells.

Similarly, cells cultured without estrogenic stimuli did not show oocytedevelopment in day 5 culture. However, 12 day cultures of ovarian cellsshowed the presence of oocytes. It is conceived that natural estrogenicstimulation is provided by fibroblasts that accompany OSE cells in theculture.

Example 1g 36 Year Old Woman

Ovarian tissue was collected from a 36 year-old woman.Immunohistochemistry of the tissue showed the presence of CK+OSE cellsand no primary follicles. New oocytes developed in the culture.

Example 1h 50 Year Old Woman

Ovarian tissue was collected from a 50 year-old woman.Immunohistochemistry of the tissue showed no OSE or primary follicles.No new oocytes developed in the culture.

Example 1i 39 Year Old Woman

Ovarian tissue was collected from a 39 year-old woman.Immunohistochemistry of the tissue showed the presence of OSE cells andCK+ mesenchymal cells in the TA, and a lack of primary follicles. Newoocytes developed in the culture.

Example 1j 55 Year Old Woman

Ovarian tissue was collected from a 55 year-old woman.Immunohistochemistry of the tissue showed no primary follicles, but CK+TA and OSE at the ovarian surface.

The ovaries also showed cortical epithelial crypts with CK+ OSE. Cultureof this tissue produced new oocytes.

Example 1k 67 Year Old Woman

Ovarian tissue was collected from a 67 year-old woman. This woman wasthe most advanced age studied. Culture of the tissue as described inExample 2 produced new oocytes. In addition, tissue from the culture wassecondarily cultured. The secondary culture contained only epithelialclusters and fibroblasts and had no granulosa cells, which were presentin the primary culture. The secondary culture of the atrophic ovariantissue of this woman produced new oocytes.

Example 2 Fertilization of Cultured Ovarian Stem Cells Derived fromInfertile Woman with Premature Ovarian Failure Example 2a Collection ofOvarian Tissue

Ovarian tissue was collected during diagnostic laparoscopic examinationunder general anesthesia from three infertile women, ages 30 (Patient1), 38 (Patient 2), and 40 (Patient 3) years old, with premature ovarianfailure (POF). First the surface of each ovary was gently scrapped withlaparoscopic scissors and cells collected by washing branches of thescissors in tissue culture medium. Additional cells were similarlycollected by laparoscopic brushes. Next each ovary was washed with 10 mlsaline solution, and the fluid was collected from the pouch of Douglas.Finally, a small biopsy (15×5 mm) was collected from each ovary and thebiopsy site was closed with a single suture. Examination of the ovariesfrom the three women revealed the absence of oocytes.

Example 2b Culture of Ovarian Tissue

All chemicals were purchased from Sigma, St. Louis, Mo., USA, exceptwhere indicated otherwise. For cultures, Dulbecco's Modified Eagle'sMedium Nutrient Mixture/F-12 Ham Medium (1:1) with L-glutamine, 15 mMHEPES, and phenol red was used. The medium was supplemented with sodiumbicarbonate—3.7 g/L, antibiotics—50 μg/ml gentamycin, 100 U/mlpenicillin, and 100 μg/ml streptomycin (DMEM/F12), and with 20%comprehensively heat inactivated human serum (HuS) of the correspondingpatient.

For this purpose, 30 ml of venous blood was collected into sterile tubesand allowed to coagulate at room temperature. After coagulation theblood sample was spun down for 10 minutes at 3000× g, serum pipettedinto new tubes and heat-inactivated for 60 minutes in the 59° C. waterbath. The serum after such extensive heat inactivation was spun downagain to sediment additionally coagulated proteins, and noncoagulatedproportion pipetted into new tubes and stored at −20° C. untilutilization.

Half of each ovarian biopsy was placed in a sterile Petri dish withDMEM/F12 medium, and a sterile surgery blade was used to scratch thecells from all surfaces. In addition, the tissue in medium was mincedwith the blade into small pieces. Next the cells were collected bypassing the medium with cells and tissue remnants through the 70 μmnylon cell strainer (BD Falcon, Bedford, Mass., USA). Cell suspensionwas spun down (1000× g, 10 minutes), supernatant removed, and cellsdiluted with DMEM/F12 supplemented with 20% HuS, and 350 μl of cellsuspension were put in each well of a four-well sterile plate (day 0).

The cell suspension collected from the pouch of Douglas was also spundown and cells similarly re-suspended and seeded. Cells were incubatedat 37° C. and 5% CO₂ (HeraCell, Haereues, Germany).

After 24 hours (day 1), the medium with unattached cells was collected,and each well gently washed with DMEM/F12 to remove majority oferythrocytes prior to adding new medium with HuS. The collectedsuspension with unattached cells was spun down, re-suspended in newmedium with HuS and seeded in additional new wells. Such cells wereassigned as “supernatants,” and remaining erythrocytes were similarlywashed next day (day 2). The cultures were evaluated daily under theinverted microscope with heated stage (Nikon, Japan).

Example 2c In vitro Maturation Medium

On day 3 of culture DMEM/F12 medium with HuS in each well was replacedwith 350 μl of in vitro Maturation Medium (MediCult IVM, Copenhagen,Denmark) supplemented with FSH and human chorionic gonadotropin (hCG).One ampule of Gonal F (Serono, Switzerland) containing 75 IU (5.5 μg)FSH was first added to 10 ml MediCult IVM medium (FSH solution). Oneampule of Pregnyl (Organon, Oss, The Netherlands) containing 5000 IU hCGwas also first added to another 10 ml of IVM Medium (hCG solution).Final IVM medium was prepared as 9 ml IVM Medium +1 ml heat inactivatedpatient's serum +100 μl FSH solution +100 μl hCG solution. The finalconcentration of hormones was 75 mlU/ml FSH +5 IU/ml hCG.

Example 2d Sperm Preparation and Utilization

On day 3 sperm was prepared on a PureSperm (Nidacon, Goteborg, Sweden)concentration gradient (80%/40%) by centrifugation at 1200 rpm perminute. After that, the 80% fraction with sperm was washed in 5 ml ofSperm Preparation Medium (MediCult, Jyllinge, Denmark) with 50 mMantioxidant hypotaurine and centrifugation at 1400 rpm per minute. Thepellet was re-suspended in 0.5 ml Sperm Preparation Medium withhypotaurine. Sperm suspension was left in a CO₂-incubator at 37° C. for30 minutes for “swim-up.” Then the aliquot of sperm suspension from thetop was taken and 5 drops of sperm suspension were put in each well withovarian cell culture, except some wells representing controls forcomparison of the sperm effect.

Example 2e Evaluation and Freezing of Embryo- and Blastocyst-LikeStructures

On days 4-6, the ovarian cell cultures were evaluated for the presenceof embryo-, morula, and blastocystlike structures. Several cell stageembryo-like structures, morulae, preblastocyst- and blastocyst-likestructures were detected. All these structures were collected and frozenand kept in a liquid nitrogen at −196° C. to be later geneticallyanalyzed and transferred in to the uterus.

Example 2f Immunohistochemistry

The remaining half of each ovarian biopsy was formalin-fixed, embeddedin paraffin, and 10 μm sections were collected on microscope slides.Sections were deparaffinized and rehydrated by immersion of slides inthe 0.01 M citrate buffer, pH 6.0, at 98° C. for 40 minutes. Slides werethen cooled to room temperature, incubated 20 minutes with mousemonoclonal antibody against high molecular weight cytokeratin, clone34βE12 (Dako, Glostrup, Denmark) diluted 1:200 in phosphate-bufferedsaline, washed, incubated with peroxidase-coupled rabbit anti-mouseimmunoglobulins (Dako), and peroxidase visualized by diaminobenzidinesolution as recommended by the vendor (Dako). Finally, the slides weredehydrated and mounted in Canada balsam. The sections were evaluatedunder the light microscope for the presence of OSE cells and granulosacells of primary follicles exhibiting brown staining for cytokeratin.

Example 2g Ovarian Surface Epithelium

Staining of biopsy sections for cytokeratin revealed the presence of anOSE, layer in ovarian biopsies from Patient 1 and from Patient 3. No OSEwas observed in ovarian biopsy from Patient 2. No primary follicles werefound in the biopsies from any of the patients.

Example 2h Results of Ovarian Stem Cell Culture

Observations of day 1 revealed sheets of OSE stem cells in Patient 1 andPatient 3 cultures, but no such sheets were observed in cultures fromPatient 2. This correlated with the lack of OSE cells in Patient 2ovarian biopsies and also with the history of autoimmune oophoritis inthis patient. In day 2 cultures of Patients 1 and 3, ovarian epithelialcells and fibroblasts attached to the bottom of the dish and very richovarian cell cultures developed. Cultures from Patient 2 ovaries showedoccasional fibroblasts only.

Primary ovarian cultures exhibited rounded stem cells and fibroblasts.Round oocyte-like cells were observed on day 3 in cultures from Patients1 and 3, showing germinal vesicle. The most developed oocyte-like cellsshowed cytoplasmic connection with smaller epithelial cells, whichappeared to support the oocyte development by providing additionalorganelles and then degenerated. Such cells appeared to originate froman incomplete division (lack of separation) of the oocyte precursor,followed by a dominance of one of the divided cells (master and slaveprinciple). In addition, most oocyte-like cells were associated withfibroblasts.

Example 2i Development of Embryo-Like Structures After Fertilization

When sperm were added to the culture, they associated with ovariancells. This resulted in creation of embryo-like structures in Patients 1and 3. These structures appeared only in fertilized cultures and not incontrol dishes without sperm.

In the Patient 1 culture, embryo-like structures appeared in the wellswith cells collected from the ovarian surface and pouch of the Douglas,and not in the culture derived from ovarian biopsies, whereas in thePatient 3, the embryo-like structures developed in culture derived fromthe ovarian biopsy.

Several hours after fertilization of OSE culture on day 3, two-cellstructures appeared which resembled two-cell embryos. This phenomenonwas observed in cultures from both Patients 1 and 3. On day 4, somefour-cell and cleavage stage embryo-like structures were apparent.

On day 5 morula-, preblastocyst-(morulae with early formation ofblastocoele), and blastocyst-like structures were observed in thecultures. These embryo-like structures detached spontaneously from thedish bottom and were present in the medium above the attached cells. Itwas possible to collect them by a sterile glass pipette and to transferthem into the fresh medium for embryo culture.

In Patient 1, there were many (about 20) morula- and preblastocyst-likestructures. The morula- and preblastocyst-like structures were smallerthan a normal blastocyst and showed no zona pellucida. In Patient 3,only one blastocyst-like structure was found. It had a volume of anormal blastocyst, contained an inner cell mass, trophectoderm, andblastocoele, but showed no zona pellucida, and appeared to hatch duringthe culture. All collected structures were frozen, each in a separatestraw.

Occasionally, parthenogenetically developed morulae were observed, butthey appeared earlier (prior to fertilization) than the embryo-likestructures developing after fertilization, and they remained attached tothe bottom of the dish and surrounded by fibroblasts. Duringcontinuation of the culture they did not detach but degenerated.

Example 3 Transfer of Embryos Into A Uterus Example 3a Preparation ofEndometrium

In women without menstrual cycles, such as women with POF, endometrialpreparation for embryo transfer is modeled on the natural menstrualcycle, using estrogen and progesterone. The initial estrogenic phase ismaintained by using daily oral estradiol 4-8 mg. The length ofestrogenic exposure is 12-14 days. Progesterone administration follows,100 mg intramuscularly daily, along with continuation of estrogensupplementation as above. Fresh or frozen-thawed embryos are transferredat the optimal time for embryo transfer, which is 2-4 days afterprogesterone initiation. Progesterone (and estrogen) administration arediscontinued once the placenta has established adequate steroidogenesis,which occurs at 7-9 week of gestation. Clinically, concentration ofserum progesterone is monitored weekly for 10 weeks after embryotransfer, when a serum level of ≧30 ng/ml typically is attained. At thatpoint, supplementation with steroids is terminated.

In patients with a regular menstrual pattern, fresh or frozen-thawedembryos are transferred into the uterus 4 days after the disappearanceof the dominant follicle in the natural cycle. Follicular growthmonitoring is performed by determination of serum 1 estradiol levels,vaginal ultrasound measurement of follicles and endometrium, and byquick urinary LH determination according to the established protocol forIVF embryo transfer in the natural cycle. After a positive urinary LHand a decrease in estradiol level, the disappearance of the dominantfollicle is observed on ultrasound.

In women with irregular menstrual cycles, a minimal stimulation with 75IU of FSH daily is started on day 7 of the menstrual cycle. When thecriteria for follicular maturity are achieved, and if the urinary LH isstill negative, ovulation is induced with 5000 IU of hCG, and thetransfer of fresh or frozen-thawed embryos is performed 6 days after hCGadministration. If the urinary LH is positive, the transfer is performedafter the disappearance of the dominant follicle.

Example 3b Transfer of Embryos

Fresh and frozen-thawed embryos are implanted into the uterus using atransfer catheter after endometrium preparation as described above.Frozen embryos are thawed using a two-step thawing protocol with thawingsolutions free of cryoprotectant. On the day of transfer, embryos arethawed at room temperature. They are transferred to two thawingsolutions free of cryoprotectant glycerol: thawing solution 1 composedof 0.5 M sucrose in the Universal IVF Medium (Medi-Cult, Jyllinge,Denmark) and thawing solution 2 composed of 0.2 M sucrose in theUniversal IVF Medium. Embryos are exposed to each solution for 10minutes at room temperature under a regulated 5% CO₂ stream (glasshood). Then they are washed and transferred into the preincubated freshUniversal IVF Medium. One or two embryos are transferred into the uterusabout 1 hour after thawing.

Further modifications, uses, and applications of the invention describedherein will be apparent to those skilled in the art. It is intended thatsuch modifications be encompassed in the claims that follow.

1. A method for obtaining a fertilized human oocyte comprising culturingin a culture medium ovarian cells including surface epithelial cells andinitially containing no oocytes, permitting the cells to remain in theculture medium for a period of time sufficient for the cells to developinto oocytes, adding sperm to the culture, permitting the sperm toassociate with the ovarian cells, resulting in one or more fertilizedoocytes.
 2. The method of claim 1 wherein the culture medium contains asource of estrogen.
 3. The method of claim 2 wherein the source ofestrogen is a chemical compound having estrogenic activity.
 4. Themethod of claim 3 wherein the cells arc permitted to remain in theculture medium for at least 4 to 6 days.
 5. The method of claim 2wherein the source of estrogen is from ovarian cells in the culture thatproduce estrogen.
 6. The method of claim 5 wherein the cells arepermitted to remain in the culture medium for at least 10 days.
 7. Themethod of claim I wherein the ovarian cells that are cultured wereobtained from a postmenopausal or anovulatory ovary.
 8. The method ofclaim 1 which further comprises permitting the fertilized oocytes todevelop into one or more embryos.
 9. The method of claim 8 which furthercomprises transferring one or more of the embryos into the uterus of anadult human female.